Project Summary Although behavior is largely structured by sensory cues, animals also benefit from random actions. Stochastic actions and variability are important for many behaviors including motor learning, search, and predator evasion. Furthermore, understanding the cellular basis of stochasticity may be pertinent to many neurological disorders involving the inability to either suppress or generate involuntary actions, including Tourette's syndrome, Huntington's chorea, and Parkinson's disease. Despite its importance, the mechanisms underlying the generation of random actions at the molecular, cellular, or network levels are poorly understood. Understanding spontaneous processes in the nervous system will benefit from research in a tractable genetic model in which both its functional role and mechanistic basis could be studied vertically across phenomenological levels. Recently, a genetically identified neuron has been identified cell (called DNa04) in the fruit fly, Drosophila that serves as a command neuron for rapid spontaneous turns, called saccades. This is a rare case of a single genetically identified neuron whose activity is necessary and sufficient for stochastic actions. Although it is already possible to record from this command neuron using 2-photon imaging and in vivo patch clamp, the primary goal of this proposal is to identify members of the upstream network that is responsible for generating its stochastic activity. Toward these goals, Specific Aim 1 of this proposal will focus on screening a collection of ~20 selective split-GAL4 lines labeling interneurons in the Lateral Accessory Lobe (LAL), the upstream neuropil region that is thought responsible for generating the spontaneous activity in the DNa04 cell. We will perform 2- photon functional imaging in intact flying flies to record from LAL interneurons and use genetic tracing techniques including trans-Tango to map the connectivity within the network. In Specific Aim 2 we will explore the function of individual cells in generating the pattern of spontaneous activity by manipulating cell physiology using a variety approaches including optogenetic activation and silencing. Specific Aim 3 will focus on psychophysical experiments aimed at quantifying and modeling the influence of sensory stimuli that modulate the frequency of spontaneous saccades. By mapping the network and experimentally manipulating the physiology of its member cells, we will lay the foundation for developing a quantitative model of the cellular basis of spontaneous actions in the brain.